Albanian j. agric. sci. 2017; (Special edition)
Agricultural University of Tirana
(Open Access)
RESEARCH ARTICLE
The identification of Tularemia agent in tissues of wild hare flesh with application of immunohistochemical avidin-biotin complex method (IMHC-ABC) BESNIK ELEZI1*, KASTRIOT KORRO2 1
PhD candidate, Faculty of Veterinary Medicine, Agricultural University of Tirana Agricultural University of Tirana
2
Faculty of Veterinary Medicine, Agricultural University of Tirana Agricultural University of Tirana
*Corresponding author; E-mail:
[email protected]
Abstract The study presents the results achieved from the application of the immunohistochemical avidin-biotin complex method (IMHC-ABC) or the assessment of the risk of the presence of the Tularemia antigen in the flesh of hares haunted by the hunters in some of the regions of Macedonia, the ex-republic of Yugoslavia. The study indicates the method applied in 70 samples taken from the hares killed by the hunters, as well as some samples taken from the restaurants which offer in their menu. The study results with the identification of 4 positive cases with Tularemia from the samples submitted to this check. The check is significant, as it presents a method of assessment of the risk of the meat of the hares with the presence of this zoonotic disease, as well as it exposes a method which has not been applied before for this disease in the region. Keywords: Immunohistochemical, avidin-biotin method, tularemia, FYROM-Macedonia, IMHC.
1. Introduction method [3, 4, 5]. Tularemia is found at least 110 Tularemia is a cyclic infection which affects the
different kinds of animals, 25 kinds of birds and a few
animals and the humans [2, 3, 4, 5]. The people get
kinds of fish. It is found in hard-bodied ticks of the
the infection from the animals with bacteremia and
genus Ixodes, Dermacentor, Haemophysalis species,
toxicemia which is often developed in very severe
etc, it is also seen in arthropods, in mites and in the
form. The infection is caused by Francisella
flies [1, 10]. They find shelter in the mites, and fleas
tularensis. In the year 1999, in Kosovo were observed
and they get transported. Rodents, such as the water
about 250 cases with tularemia in humans [1]. The
voles (Arvicola terrestris), hares etc, as well as some
infection was diagnosed in the Institute of Veterinary
carnivore insects, are very sensitive towards the
Research “Bilal Golemi” in Tiranë through the
infection and they are severely infected [3, 4, 9].
serological test [1, 3, 9, 10] . This was the first case of
Biovar A is met at lagomorphs, whereas holoarctic
infection diagnosis in our country in humans. During
biovar B is met mostly at small rodents. Furthermore
this period, in our country studies were made in
sensitive but less affected are the field rats (Microbus
humans who were suspected for its presence. From
arvalis, Apodemus-Arten), hamsters, hares, eagles, etc
these checks the infection was diagnosed only in Hasi
and from the domesticated animals domestic sheep
region. Since from the year 1999, in Albania are not
and hares. Perissodactyls as well as carnivores are less
observed suspicious cases of infection either in
sensitive, but they get affected more than minks and
humans or in wild rodents [3, 7, 8]. From our study
foxes [3, 8]. The animals with very high sensitivity
during the years 2015-2016 for the diagnosis of
are the 1st grade reservoirs and epizootic before all,
Tularemia in wild hares of the western part of
contagious for the humans and they create infective
Macedonia, we have identified 47 positive cases for
foci [1, 5, 7].
the presence of tularemia b means of the serological
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Elezi & Korro 2017
2. Material and Methods
Dako, Glostrup, Denmark) [3, 4, 6, 7]. A series of lesions were incubated with phosphate buffer solution
For the realization of this study were taken 70 samples
and it will be used as a negative control [3, 7]. We
from liver, lymph nodes and the lung of the
used the immunohistochemical avidin-biotin complex
postmortem hares [3, 7].
method (IMHC-ABC) which was realized on the
2.1.
The
immunohistochemical
staining
formalin-fixed tissues respecting the association protocol of the kit [2, 6].
Avidin-Biotin Complex (IHC- ABC) method The immunohistochemical avidin-biotin complex method
(IMHC-ABC),
was
applied
for
the
demonstration of the lipopolysaccharide antigen of F.
3. Results and Discussion
tularensis in tissue sections. Immediately after the antigen deparafinisation and extraction (in an
During our research 70 samples were collected from
microwave oven in 750 W for 20 minutes in citrate
the tissues of the hares killed by the hunters of
tampon, pH 6,0), the lesions were incubated in
Macedonia in the regions of Debresh, Nerove,
solution 3% H2O2 for 10 minutes in pH 6,0 and then
Allbance, Presille, Bellushine, Haracine, Tearce etc.,
in a solution of 2% skimmed milk for 20 minutes[3,
as well as from the shops and the restaurants which
7]. The samples were incubated during the night in
served the hare meat in their menu [1, 3, 10]. The
370 C, with a proportion 1: 6000 of the dilution of F
number of collected samples was considerable
tularensis [3, 4, 7, 8]. Lipopolysaccharide with the
considering that the population of the above areas
monoclonal-specific antibodies produced in rats
does not prefer the consummation of wild hare meat
(clones
Chemicon
[1, 3]. The collected samples were sent in the Lab of
International Inc, Southhampton, UK) the fusion of
the Wildlife Disease at the Faculty of Veterinary
antibodies were detected by means of peroxidase
Medicine, in which were realized 70 formalin-fixed
reaction and use of specific substrate, a reaction which
lesions and we followed the protocol associating the
is exposed with dark brown colour (EnVisionb Kit,
kit [1, 3, 9, 10].
FB11
and
T14,MAB8267,
Figure 1. The results acquired from this study.
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The identification of Tularemia agent in tissues of wild hare flesh with application IMHC-ABC
Figure 2. Region of samples collected.
4. Conclusions
6. References
1. Application of the (IHC- ABC) method I discover cases with presence of Tularemia antigen in the liver (1 case), in lymph node 2 cases and in the lung 1 case. 2. Application of the (IHC- ABC) method increases the detection, diagnostic sensitivity and the level of assurance for the consumer concerning the meat of the hare with origin from the wild populations. 3. The results of the method encourage the researchers to apply this method also in other studies on this disease, as an important part in the confirmation of
1. Berxholi K: Zoonozat Bakteriale, Virale dhe parazitare: Tirane; 2015. 2. DSMZ: Bacterial Nomenclature Up-toDate (Approved List,Validation List). http://www.dsmz.de/microorganisms/bact erial_ nomenclature.php. Accessed September 2009. 3. Elezi B.: Tularemia under Histological and Immunohistochemistry Examinations. Anglisticum Jurnal Mk., In press.
Tularemia antigen.
4. Friend M: Tularemia: Circular 1297. U.S. Geological Survey, Reston, VA, 2006.
5. Acknowledgements
5. Gill V, Cunha, BA.: Tularemia pneumonia. Semin Respir Infect 1997, 12:61–67.
We want to thank Associate of Hunters of FYROM Macedonia and staff of Wildlife lab in Faculty of Veterinary Medicine, Agricultural University of Tirana.
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6. Greco D, Allegrini G, Tizzi T, Ninu E, Lamanna A, Luzi S: A waterborne tularemia outbreak. Eur J Epidemiol 3:35–38, 1987.
Elezi & Korro 2017
7. Gyuranecz M, Szeredi L, L. Makrai L, Fodor A, Meszaros R, Szepe B, Fuleki M, and. Erdelyi K: Tularemia of European Brown Hare (Lepus europaeus): A Pathological, Histopathological, and Immunohistochemical Study. Veterinary Pathology 47(5) 958-963 http://vet.sagepub.com 8. Gyuranecz M, Fodor L, Makrai L, Szoke I, Janosi K, Krisztalovics K, Erdelyi K: Generalized tularemia in a vervet
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monkey (Chlorocebus aethiops) and a patas monkey (Erythrocebus patas) in a zoo. J Vet Diagn Invest 21:384–387, 2009. 9. Koleci Xh: Semundjet infektive: Tirane; 2009. 10. Korro K, Keci R, Berxholi K: Semundjet e kafsheve te Egra: Tirane; 2013.
